Multiparametric Biomechanical and Biochemical Phenotypic Profiling of Single Cancer Cells Using an Elasticity Microcytometer

Deep phenotyping of single cancer cells is of critical importance in the era of precision medicine to advance understanding of relationships between gene mutation and cell phenotype and to elucidate the biological nature of tumor heterogeneity. Existing microfluidic single‐cell phenotyping tools, however, are limited to phenotypic measurements of 1–2 selected morphological and physiological features of single cells. Herein a microfluidic elasticity microcytometer is reported for multiparametric biomechanical and biochemical phenotypic profiling of free‐floating, live single cancer cells for quantitative, simultaneous characterizations of cell size, cell deformability/stiffness, and surface receptors. The elasticity microcytometer is implemented for measurements and comparisons of four human cell lines with distinct metastatic potentials and derived from different human tissues. An analytical model is developed from first principles for the first time to convert cell deformation and adhesion information of single cancer cells encapsulated inside the elasticity microcytometer to cell deformability/stiffness and surface protein expression. Together, the elasticity microcytometer holds great promise for comprehensive molecular, cellular, and biomechanical phenotypic profiling of live cancer cells at the single cell level, critical for studying intratumor cellular and molecular heterogeneity using low‐abundance, clinically relevant human cancer cells.     

Effect of Hydroxyapatite Nanorods on the Fate of Human Adipose‐Derived Stem Cells Assessed In Situ at the Single Cell Level with a High‐Throughput,Real‐Time Microfluidic Chip

The fate of stem cells at the single cell level with limited communication with other cells is still unknown due to the lack of an efficient tool for highly accurate molecular detection. Moreover, the conditional sensitivity of biological experiments requires a sufficient number of parallel experiments to support a conclusion. In this work, a microfluidic single cell chip is designed for use with a protein chip to investigate the effect of hydroxyapatite (HAp) on the osteogenic differentiation of human adipose‐derived stem cells (hADSCs) in situ at the single cell level. By successfully detecting secretory proteins in situ, it is found that the HAp nanorods enhance osteogenic differentiation at the single cell level. In the chip, the single cell seeding approach confirms the osteogenic differentiation of the hADSCs, which endocytoses HAp, by reducing the influence of the factors secreted by neighboring differentiating cells. Most importantly, more than 7000 microchambers provide a sufficient number of parallel experiments for statistical analysis, which ensure a high level of repeatability of the HAp nanorod‐induced osteogenic differentiation. The microfluidic chip comprising single cell culture microchambers with in situ detection capability is a promising tool for research on cell behavior or cell fate at the single cell level.     

A SERS‐Assisted 3D Barcode Chip for High‐Throughput Biosensing

A surface enhanced Raman scattering (SERS)‐assisted 3D barcode chip has been developed for high‐throughput biosensing. The 3D barcode is realized through joint 2D spatial encoding with the Raman spectroscopic encoding, which stores the SERS fingerprint information in the format of a 2D array. Here, the concept of SERS‐assisted 3D barcode is demonstrated through multiplex immunoassay, where simultaneous detection of multiple targets in different samples has been achieved using a microfluidic platform. First, multiple proteins in different samples are spatially separated using a microfluidic patterned antibody barcode substrate, forming a 2D hybridization array. Then the SERS probes are used to identify and quantify the proteins. As different SERS probes are labeled with different Raman reporters, they could be employed as “SERS tags” to incorporate spectroscopic information into the 3D barcode. In this 3D barcode, the 2D spatial information helps to differentiate the samples and targets while the SERS information allows quantitative multiplex detection. It is found that the SERS‐assisted 3D barcode chip can not only accomplish one‐step multiplex detection within 30 min but also achieve an ultrasensitivity down to 10 fg mL^?1 (≈70 aM), which is expected to provide a promising tool for high‐throughput biomedical applications.     

In Situ Two‐Step Photoreduced SERS Materials for On‐Chip Single‐Molecule Spectroscopy with High Reproducibility

A method is developed to synthesize surface‐enhanced Raman scattering (SERS) materials capable of single‐molecule detection, integrated with a microfluidic system. Using a focused laser, silver nanoparticle aggregates as SERS monitors are fabricated in a microfluidic channel through photochemical reduction. After washing out the monitor, the aggregates are irradiated again by the same laser. This key step leads to full reduction of the residual reactants, which generates numerous small silver nanoparticles on the former nanoaggregates. Consequently, the enhancement ability of the SERS monitor is greatly boosted due to the emergence of new “hot spots.” At the same time, the influence of the notorious “memory effect” in microfluidics is substantially suppressed due to the depletion of surface residues. Taking these advantages, two‐step photoreduced SERS materials are able to detect different types of molecules with the concentration down to 10^?13m . Based on a well‐accepted bianalyte approach, it is proved that the detection limit reaches the single‐molecule level. From a practical point of view, the detection reproducibility at different probing concentrations is also investigated. It is found that the effective single‐molecule SERS measurements can be raised up to ≈50%. This microfluidic SERS with high reproducibility and ultrasensitivity will find promising applications in on‐chip single‐molecule spectroscopy.     

Tracking Drug‐Induced Epithelial–Mesenchymal Transition in Breast Cancer by a Microfluidic Surface‐Enhanced Raman Spectroscopy Immunoassay

Epithelial–mesenchymal transition (EMT) is a primary mechanism for cancer metastasis. Detecting the activation of EMT can potentially convey signs of metastasis to guide treatment management and improve patient survival. One of the classic signatures of EMT is characterized by dynamic changes in cellular expression levels of E‐cadherin and N‐cadherin, whose soluble active fragments have recently been reported to be biomarkers for cancer diagnosis and prognosis. Herein, a microfluidic immunoassay (termed “SERS immunoassay”) based on sensitive and simultaneous detection of soluble E‐cadherin (sE‐cadherin) and soluble N‐cadherin (sN‐cadherin) for EMT monitoring in patients' plasma is presented. The SERS immunoassay integrates in situ nanomixing and surface‐enhanced Raman scattering readout to enable accurate detection of sE‐cadherin and sN‐cadherin from as low as 10 cells mL^?1. This assay enables tracking of a concurrent decrease in sE‐cadherin and increase in sN‐cadherin in breast cancer cells undergoing drug‐induced mesenchymal transformation. The clinical potential of the SERS immunoassay is further demonstrated by successful detection of sE‐cadherin and sN‐cadherin in metastatic stage IV breast cancer patient plasma samples. The SERS immunoassay can potentially sense the activation of EMT to provide early indications of cancer invasions or metastasis.     

<Emphasis Type="Italic">In situ</Emphasis> probing of cell–cell communications with surface-enhanced Raman scattering (SERS) nanoprobes and microfluidic networks for screening of immunotherapeutic drugs

Discovering novel drugs for cancer immunotherapy requires a robust in vitro drug screening platform that allows for straightforward probing of cell-cell communications.Here,we combined surface-enhanced Raman scattering (SERS) nanoprobes with microfluidic networks to monitor in situ the cancer-immune system intercellular communications.The microfluidic platform links up immune cells with cancer cells,where the cancer-cell secretions act as signaling mediators.First,gold@silver core-shell nanorods were employed to fabricate SERS immunoprobes for analysis of the signaling molecules.Multiple cancer secretions in a tumor microenvironment were quantitatively analyzed by a SERS-assisted three-dimensional (3D) barcode immunoassay with high sensitivity (1 ng/mL).Second,in an on-chip cell proliferation assay,multiple immunosuppressive proteins secreted by cancer cells were found to inhibit activation of immune cells,indicating that the platform simulates the physiological process of cancer-immune system communications.Furthermore,potential drug candidates were tested on this platform.A quantitative SERS immunoassay was performed to evaluate drug efficacy at regulating the secretion behavior of cancer cells and the activity of immune cells.This assay showed the suitability of this platform for in vitro drug screening.It is expected that the fully integrated and highly automated SERS-microfluidic platform will become a powerful analytical tool for probing intercellular communications and should accelerate the discovery and clinical validation of novel drugs.     

Integrative Magneto-Microfluidic Separation of Immune Cells Facilitates Clinical Functional Assays

Accurately analyzing the functional activities of natural killer (NK) cells in clinical diagnosis remains challenging due to their coupling with other immune effectors. To address this, an integrated immune cell separator is required, which necessitates a streamlined sample preparation workflow including immunological cell isolation, removal of excess red blood cells (RBCs), and buffer exchange for downstream analysis. Here, a self-powered integrated magneto-microfluidic cell separation (SMS) chip is presented, which outputs high-purity target immune cells by simply inputting whole blood. The SMS chip intensifies the magnetic field gradient using an iron sphere-filled inlet reservoir for high-performance immuno-magnetic cell selection and separates target cells size-selectively using a microfluidic lattice for RBC removal and buffer exchange. In addition, the chip incorporates self-powered microfluidic pumping through a degassed polydimethylsiloxane chip, enabling the rapid isolation of NK cells at the place of blood collection within 40 min. This chip is used to isolate NK cells from whole blood samples of hepatocellular cancer patients and healthy volunteers and examined their functional activities to identify potential abnormalities in NK cell function. The SMS chip is simple to use, rapid to sort, and requires small blood volumes, thus facilitating the use of immune cell subtypes for cell-based diagnosis.     

Selective Capture and Quick Detection of Targeting Cells with SERS‐Coding Microsphere Suspension Chip

Circulating tumor cells (CTCs) captured from blood fluid represent recurrent cancers and metastatic lesions to monitor the situation of cancers. We develop surface‐enhanced Raman scattering (SERS)‐coding microsphere suspension chip as a new strategy for fast and efficient capture, recovery, and detection of targeting cancer cells. Using HeLa cells as model CTCs, we first utilize folate as a recognition molecule to be immobilized in magnetic composite microspheres for capturing HeLa cells and attaining high capturing efficacy (up to 95%). After capturing cells, the composite microsphere, which utilizes a disulfide bond as crosslinker in the polymer shell and as a spacer for linking folate, can recycle 90% cells within 20 min eluted by glutathion solution. Taking advantage of the SERS with fingerprint features, we characterize captured/recovered cells with the unique signal of report‐molecule 4‐aminothiophenol through introducing the SERS‐coding microsphere suspension chip to CTCs. Finally, the exploratory experiment of sieving cells shows that the magnetic composite microspheres can selectively capture the HeLa cells from samples of mixed cells, indicating that these magnetic composite microspheres have potential in real blood samples for capturing CTCs.     

Multifunctional Silver‐Embedded Magnetic Nanoparticles as SERS Nanoprobes and Their Applications

In this study, surface‐enhanced Raman spectroscopy (SERS)‐encoded magnetic nanoparticles (NPs) are prepared and utilized as a multifunctional tagging material for cancer‐cell targeting and separation. First, silver‐embedded magnetic NPs are prepared, composed of an 18‐nm magnetic core and a 16‐nm‐thick silica shell with silver NPs formed on the surface. After simple aromatic compounds are adsorbed on the silver‐embedded magnetic NPs, they are coated with silica to provide them with chemical and physical stability. The resulting silica‐encapsulated magnetic NPs (M‐SERS dots) produce strong SERS signals and have magnetic properties. In a model application as a tagging material, the M‐SERS dots are successfully utilized for targeting breast‐cancer cells (SKBR3) and floating leukemia cells (SP2/O). The targeted cancer cells can be easily separated from the untargeted cells using an external magnetic field. The separated targeted cancer cells exhibit a Raman signal originating from the M‐SERS dots. This system proves to be an efficient tool for separating targeted cells. Additionally, the magnetic‐field‐induced hot spots, which can provide a 1000‐times‐stronger SERS intensity due to aggregation of the NPs, are studied.     

Screening Therapeutic Agents Specific to Breast Cancer Stem Cells Using a Microfluidic Single‐Cell Clone‐Forming Inhibition Assay

Screens of cancer stem cells (CSCs)‐specific agents present significant challenges to conventional cell assays due to the difficulty in preparing CSCs ready for drug testing. To overcome this limitation, developed is a microfluidic single‐cell assay for screening breast cancer stem cell–specific agents. This assay takes advantage of the single‐cell clone‐forming capability of CSCs, which can be specifically inhibited by CSC‐targeting agents. The single‐cell assay is performed on a microfluidic chip with an array of 3840 cell‐capturing units; the single‐cell arrays are easily formed by flowing a cell suspension into the microchip. Achieved is a single cell‐capture rate of ≈60% thus allowing more than 2000 single cells to be analyzed in a single test. Over long‐term suspension culture, only a minority of cells survive and form tumorspheres. The clone‐formation rate of MCF‐7, MDA‐MB‐231, and T47D cells is 1.67%, 5.78%, and 5.24%, respectively. The clone‐forming inhibition assay is conducted by exposing the single‐cell arrays to a set of anticancer agents. The CSC‐targeting agents show complete inhibition of single‐cell clone formation while the nontargeting ones show incomplete inhibition effects. The resulting microfluidic single‐cell assay with the potential to screen CSC‐specific agents with high efficiency provides new tools for individualized tumor therapy.     

Nanoelectromechanical Chip (NELMEC) Combination of Nanoelectronics and Microfluidics to Diagnose Epithelial and Mesenchymal Circulating Tumor Cells from Leukocytes

An integrated nano‐electromechanical chip (NELMEC) has been developed for the label‐free distinguishing of both epithelial and mesenchymal circulating tumor cells (ECTCs and MCTCs, respectively) from white blood cells (WBCs). This nanoelectronic microfluidic chip fabricated by silicon micromachining can trap large single cells (>12 µm) at the opening of the analysis microchannel arrays. The nature of the captured cells is detected using silicon nanograss (SiNG) electrodes patterned at the entrance of the channels. There is an observable difference between the membrane capacitance of the ECTCs and MCTCs and that of WBCs (measured using SiNG electrodes), which is the key indication for our diagnosis. The NELMEC chip not only solves the problem of the size overlap between CTCs and WBCs but also detects MCTCs without the need for any markers or tagging processes, which has been an important problem in previously reported CTC detection systems. The great conductivity of the gold‐coated SiNG nanocontacts as well as their safe penetration into the membrane of captured cells, facilitate a precise and direct signal extraction to distinguish the type of captured cell. The results achieved from epithelial (MCF‐7) and mesenchymal (MDA‐MB231) breast cancer cells circulated in unprocessed blood suggest the significant applications for these diagnostic abilities of NELMEC.     

On-chip transformation of bacteria

On-chip transformation of Escherichia coli cells was accomplished for the first time using a microbial array chip. The continuous E. coli transformation procedures were performed on a chip in which the microcompartment was composed of PDMS microfluidic channels and a silicon substrate predeposited with different plasmid DNAs. The PDMS microfluidic device enabled the parallel transformation of E. coli cells with various plasmid DNAs by separating each transformation area. The phenotypic differences reflecting different plasmid DNAs were identified by various approaches such as colorimetry, fluorometry, and electrochemical methods. This microbial array chip could become a versatile tool for many cell biological applications.     

Tape‐Imprinted Hierarchical Lotus Seedpod‐Like Arrays for Extraordinary Surface‐Enhanced Raman Spectroscopy

A novel two‐side‐activatable high‐performance surface‐enhanced Raman spectroscopy (SERS) substrate is developed based on the tape‐imprinting method. It features 3D full‐space‐distributed hot spots originating from the hierarchical lotus seedpod‐like silver arrays, which offer ultrasensitive, uniform, reproducible, and reliable quantitative measurements with an inherent internal standard. This excellent SERS substrate also holds great promise in practical in situ molecule detection on curved surfaces, such as pesticides on fruit, which is not yet possible with the traditional rigid or flexible material‐based SERS counterparts.     

Isolation and Profiling of Circulating Tumor‐Associated Exosomes Using Extracellular Vesicular Lipid–Protein Binding Affinity Based Microfluidic Device

Extracellular vesicles (EVs) are emerging as a potential diagnostic test for cancer. Owing to the recent advances in microfluidics, on‐chip EV isolation is showing promise with respect to improved recovery rates, smaller necessary sample volumes, and shorter processing times than ultracentrifugation. Immunoaffinity‐based microfluidic EV isolation using anti‐CD63 is widely used; however, anti‐CD63 is not specific to cancer‐EVs, and some cancers secrete EVs with low expression of CD63. Alternatively, phosphatidylserine (PS), usually expressed in the inner leaflet of the lipid bilayer of the cells, is shown to be expressed on the outer surface of cancer‐associated EVs. A new exosome isolation microfluidic device (^newExoChip), conjugated with a PS‐specific protein, to isolate cancer‐associated exosomes from plasma, is presented. The device achieves 90% capture efficiency for cancer cell exosomes compared to 38% for healthy exosomes and isolates 35% more A549‐derived exosomes than an anti‐CD63‐conjugated device. Immobilized exosomes are then easily released using Ca²⁺ chelation. The recovered exosomes from clinical samples are characterized by electron microscopy and western‐blot analysis, revealing exosomal shapes and exosomal protein expressions. The ^newExoChip facilitates the isolation of a specific subset of exosomes, allowing the exploration of the undiscovered roles of exosomes in cancer progression and metastasis.     

Transport, location, and quantal release monitoring of single cells on a microfluidic device

A novel microfluidic device has been developed for on-chip transport, location, and quantal release monitoring of single cells. The microfluidic device consists of a plate of PDMS containing channels for introducing cells and stimulants and a glass substrate into which a cell micro-chamber was etched. The two tightly reversibly sealed plates can be separated for respective cleaning, which significantly extends the lifetime of the microchip that is frequently clogged in cell analysis experiments. Using hydraulic pressure, single cells were transported and located on the microfluidic chip. After location of a single PC12 cell on the microfluidic chip, the cell was stimulated by nicotine that was also introduced through the micro-channels, and the quantum release of dopamine from the cell was amperometricly detected with our designed carbon fiber microelectrode. The results have demonstrated the convenience and efficiency of using the microfluidic chip for monitoring of quantal release from single cells and have offered a facile method for the analysis of single cells on microfluidic devices.     

Simultaneous or Sequential Orthogonal Gradient Formation in a 3D Cell Culture Microfluidic Platform

Biochemical gradients are ubiquitous in biology. At the tissue level, they dictate differentiation patterning or cell migration. Recapitulating in vitro the complexity of such concentration profiles with great spatial and dynamic control is crucial in order to understand the underlying mechanisms of biological phenomena. Here, a microfluidic design capable of generating diffusion‐driven, simultaneous or sequential, orthogonal linear concentration gradients in a 3D cell‐embedded scaffold is described. Formation and stability of the orthogonal gradients are demonstrated by computational and fluorescent dextran‐based characterizations. Then, system utility is explored in two biological systems. First, stem cells are subjected to orthogonal gradients of morphogens in order to mimic the localized differentiation of motor neurons in the neural tube. Similarly to in vivo, motor neurons preferentially differentiate in regions of high concentration of retinoic acid and smoothened agonist (acting as sonic hedgehog), in a concentration‐dependent fashion. Then, a rotating gradient is applied to HT1080 cancer cells and the change in migration direction is investigated as the cells adapt to a new chemical environment. The response time of ≈4 h is reported. These two examples demonstrate the versatility of this new design that can also prove useful in many applications including tissue engineering and drug screening.     

Microfluidic Single‐Cell Omics Analysis

The commonly existing cellular heterogeneity plays a critical role in biological processes such as embryonic development, cell differentiation, and disease progress. Single‐cell omics‐based heterogeneous studies have great significance for identifying different cell populations, discovering new cell types, revealing informative cell features, and uncovering significant interrelationships between cells. Recently, microfluidics has evolved to be a powerful technology for single‐cell omics analysis due to its merits of throughput, sensitivity, and accuracy. Herein, the recent advances of microfluidic single‐cell omics analysis, including different microfluidic platform designs, lysis strategies, and omics analysis techniques, are reviewed. Representative applications of microfluidic single‐cell omics analysis in complex biological studies are then summarized. Finally, a few perspectives on the future challenges and development trends of microfluidic‐assisted single‐cell omics analysis are discussed.     

Microfluidic‐Based Approaches in Targeted Cell/Particle Separation Based on Physical Properties: Fundamentals and Applications

Cell separation is a key step in many biomedical research areas including biotechnology, cancer research, regenerative medicine, and drug discovery. While conventional cell sorting approaches have led to high‐efficiency sorting by exploiting the cell's specific properties, microfluidics has shown great promise in cell separation by exploiting different physical principles and using different properties of the cells. In particular, label‐free cell separation techniques are highly recommended to minimize cell damage and avoid costly and labor‐intensive steps of labeling molecular signatures of cells. In general, microfluidic‐based cell sorting approaches can separate cells using “intrinsic” (e.g., fluid dynamic forces) versus “extrinsic” external forces (e.g., magnetic, electric field, etc.) and by using different properties of cells including size, density, deformability, shape, as well as electrical, magnetic, and compressibility/acoustic properties to select target cells from a heterogeneous cell population. In this work, principles and applications of the most commonly used label‐free microfluidic‐based cell separation methods are described. In particular, applications of microfluidic methods for the separation of circulating tumor cells, blood cells, immune cells, stem cells, and other biological cells are summarized. Computational approaches complementing such microfluidic methods are also explained. Finally, challenges and perspectives to further develop microfluidic‐based cell separation methods are discussed.     

Fabrication of a microfluidic chip containing dam,weirs and gradient generator for studying cellular response to chemical modulation

A microfluidic chip was designed and fabricated for studying cellular response to chemical modulation. The microfluidic network comprised an up-stream gradient-generating module and a down-stream cell culture module. The microchip was composed of a piece of glass plate and a covered PDMS film. By using a two-step wet etching method, the dam structure was fabricated on the inlet of the cell chamber facilitating cell positioning, and a series of weir structures were fabricated on the bottom of cell culture reservoirs facilitating cell seeding. This microfluidics exploited the advantage of lab-on-a-chip technology by integrating the generation of chemical concentration gradients and a series of cell operations including seeding, culture, stimulation and staining into a chip. Steady concentration gradients were generated by flowing two fluids in the network. Over time observation showed that the microchip was suitable for cell seeding and culture. The microchip described above was applied in studying the roles of As₂O₃ and buthionine sulfoximine (BSO) in mediating intracellular levels of reduced glutathione (GSH) and reactive oxygen species (ROS) in MCF-7 cells. MCF-7 cells showed dose dependent reaction to the chemical modulations. Upon the treatment with both As₂O₃ and BSO, GSH levels were down-regulated but ROS levels were up-regulated. As₂O₃ showed a stronger effect on ROS enhancement, while BSO was more effective on GSH depletion. The integrated microfluidic chip is able to perform multiparametric pharmacological profiling with easy operation, thus holds great potential for extrapolation to the cell based high-content drug screening.